cb1  (Alomone Labs)

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Identification and conservation of the axolotl endocannabinoid receptors. (A) Protein sequence alignment of the putative axolotl CB1 sequence with the rat and zebrafish CB1 sequence. (B) Protein sequence alignment of the putative axolotl CB2 sequence with the rat and zebrafish CB2 sequence. Red asterisks and red boxes indicate amino acids that are conserved between all three species. (C) Western blot using the rat CB1 antibody on axolotl tail tissue demonstrates a single prominent band at ~120 kDa ( n = 3). (D) Western blot using the rat CB2 antibody on axolotl tail tissue demonstrates two bands at a similar molecular weight of ~46 kDa ( n = 3). MW = molecular weight (for each band in ladder). (E) Preadsorption control for the CB1 antibody using either CB1 or CB2 antigenic peptides ( n = 3). (F) Preadsorption control for the CB2 antibody using CB1 or CB2 antigenic peptides ( n = 3).

Article Snippet: Overnight incubations in primary antibodies were then performed at 4°C using 1:1000 CB1 (Alomone Labs) and 1:1000 CB2 (Alomone Labs).

Techniques: Sequencing, Western Blot, Molecular Weight, Control

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: CB1 and CB2 are upregulated in response to tail amputation. (A) Western blot analysis demonstrates a significant upregulation of CB1 in the first 3 days after tail amputation, compared to uninjured controls ( n = 3; F (4,40) = 5.994, p = .0007, one‐way ANOVA). (B) No change in CB2 expression is shown in the first 3 days post tail amputation ( n = 3; F (4,40) = 2.779, p = .0397, one‐way ANOVA). (C) Western blot analysis demonstrates a significant upregulation of CB1 expression at both 7 and 14 days after tail amputation ( n = 3; F (2,24) = 15.97, p < .0001, one‐way ANOVA). (D) Western blot analysis demonstrates a significant upregulation of CB2 at 7 and 14 days post tail amputation ( n = 3; F (2,24) = 10.84, p = .0004, one‐way ANOVA). Uninj = uninjured tail tissue. hpa = hours post tail amputation; dpa = days post tail amputation. ns = not significant. * p < .05, ** p < .01, *** p < .001, *** *p < .0001 compared to uninjured controls. # p < .05.

Article Snippet: Overnight incubations in primary antibodies were then performed at 4°C using 1:1000 CB1 (Alomone Labs) and 1:1000 CB2 (Alomone Labs).

Techniques: Western Blot, Expressing

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: CB1 and CB2 are expressed in ependymoglia and neurons in the regenerating spinal cord. (A) Schematic displays the cell‐type architecture of the axolotl spinal cord. The spinal cord is comprised of ependymoglial cells (blue) that line the central canal (cc) of the spinal cord. These ependymoglia extend GFAP + processes toward the periphery of the spinal cord. The spinal cord also contains NeuN + neurons (green) that surround the ependymoglia and extend axons that express β‐III‐tubulin. (B) Immunohistochemistry ( n = 3) shows the absence of CB1 from neuronal cell bodies (iv), and shows the co‐localization of CB1 with β‐III‐tubulin in axons (viii, yellow arrow) and with GFAP in glial cell processes (xii, blue arrow). (C) Immunohistochemistry ( n = 3) shows the absence of CB2 from neuronal cell bodies (iv), and displays the co‐localization of CB2 with β‐III‐tubulin in axons (viii, yellow arrow) and with GFAP in glial cells (xii, blue arrow). (D) Fluorescent in situ hybridization ( n = 2) demonstrates cb1 mRNA expression in both neurons (yellow arrow) and ependymoglia (blue arrow). Scale bars: 100 μm.

Article Snippet: Overnight incubations in primary antibodies were then performed at 4°C using 1:1000 CB1 (Alomone Labs) and 1:1000 CB2 (Alomone Labs).

Techniques: Immunohistochemistry, In Situ Hybridization, Expressing

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Inhibiting CB1 and CB2 receptor signaling impairs tail regeneration. (A) Representative images of tail regenerates after a 7‐day treatment with the vehicle (control, i), 1 μM AM251 (ii), or 1 μM AM630 (iii). Black dotted line indicates the original plane of amputation. Scale bar: 1 mm. (B, C) Graphs show that the proportional increase in axolotl body length was significantly reduced following either a 7‐day treatment with either 1 μM AM251 ( n = 8; B) or after a 7‐day treatment with 1 μM AM630 ( n = 8; C) compared to the vehicle control (unpaired t tests). (D) Graph shows a significant reduction in the proportional increase in axolotl body length (7 days after tail amputation) following only a 1‐day pulse treatment with either 1 μM AM251 ( n = 10) or 1 μM AM630 ( n = 10), compared to vehicle controls ( n = 10; F (2,27) = 18.86; p < .0001, one‐way ANOVA). (E) Western blot analyses show that treatment with AM251 prevented the upregulation of CB1 that normally occurs in untreated or vehicle‐treated control animals at 7‐days post tail amputation ( n = 3; Constant 7‐day bath treatment: F (3,32) = 14.69; p < .0001; 1‐day pulse treatment: F (3,32) = 18.60; p < .0001; one‐way ANOVAs). Representative blot for 1‐day pulse treatment shown. (F) Treatment with AM630 prevented the upregulation of CB2 that normally occurs in untreated or vehicle‐treated control animals at 7‐days post tail amputation ( n = 3; constant treatment: F (3,32) = 24.80; p < .0001; 1‐day pulse treatment: F (3,32) = 11.60; p < .0001; one‐way ANOVAs). Representative blot for 7‐day constant treatment shown. * *p < .01, ** *p < .001, *** *p < .0001 compared to vehicle controls. ### p < .001. #### p < .0001.

Article Snippet: Overnight incubations in primary antibodies were then performed at 4°C using 1:1000 CB1 (Alomone Labs) and 1:1000 CB2 (Alomone Labs).

Techniques: Control, Western Blot

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Inhibiting cannabinoid receptor activity reduces ependymoglial cell proliferation and upregulates GFAP + in glial cell processes. (A) Representative images of EdU + cells in the regenerating axolotl spinal cord at 7‐days post tail amputation after treatment with 1 μM AM251 (ii), 1 μM AM630 (iii), or the vehicle (control, i). White dotted circles outline the spinal cord. (B) Graph shows a significant reduction in the proportion of EdU + cells in the axolotl spinal cord at 7‐days post tail amputation after treatment with either 1 μM AM251 ( n = 4) or 1 μM AM630 ( n = 4) in comparison to vehicle controls ( n = 4; F (2,9) = 25.25; p = .0002, one‐way ANOVA). ** *p < .001 compared to vehicle controls. (C) Representative images of GFAP expression in uninjured axolotl tail tissue (i) and in regenerating tail tissue (ii) at 7‐days post tail amputation (dpa). (D) Quantified western blot data demonstrates a significant reduction in GFAP expression in the first 7‐days post tail amputation in comparison to uninjured tail tissue ( n = 3; F (3,32) = 25.97, p < .0001, one‐way ANOVA). ** *p < .001 compared to uninjured controls. (E, F) Immunohistochemistry shows GFAP expression paired with either CB1 (E) or CB2 (F) staining in the axolotl spinal cord at 7‐days post tail amputation after treatment with 1 μM AM251 (Eii), or 1 μM AM630 (Fii) or the vehicle (controls, Ei and Fi). Scale bars = 100 μm.

Article Snippet: Overnight incubations in primary antibodies were then performed at 4°C using 1:1000 CB1 (Alomone Labs) and 1:1000 CB2 (Alomone Labs).

Techniques: Activity Assay, Control, Comparison, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Schematic representation of protoporphyrin IX (PpIX) generation in normal and cancer cells. Upon exogenous stimulation with 5-ALA, cells generate PpIX via the haem biosynthesis pathway. PpIX is subsequently converted to haem by FECH or transported outside the cells through efflux receptors such as ABCB1. As oncogenic transformation activates enzymes of the haem biosynthesis pathway, cancer cells generate PpIX more efficiently than normal cells. As the Ras/MEK pathway promotes PpIX conversion to haem and PpIX efflux through ABCB1, MEK inhibition further enhances PpIX accumulation in cancer cells.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Transformation Assay, Inhibition

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Oncogenic Ras/MEK regulates PpIX accumulation via ABCB1 and FECH. ( A ) Representative overlay histograms showing surface ABCB1 expression in NIH3T3 cells and RasV12 cells treated with or without different concentrations of MEK inhibitor, U0126. ( B ) Plot shows mean ± SD mean fluorescence intensity (MFI) from 3 independent experiments. ( C ) Mean ± SD relative FECH activity in NIH3T3 cells and RasV12 cells treated with or without different concentrations of U0126 from 3 independent experiments. ( D ) Representative western blot showing FECH expression and ERK phosphorylation levels in NIH3T3 and RasV12 cells treated with or without different concentrations of U0126. The relative band densities (RD) are indicated. *p < 0.01 by one-way ANOVA with Turkey's posthoc test.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Expressing, Fluorescence, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Oncogenic Ras regulates ABCB1 expression via RSKs. ( A ) Representative western blot showing RSK phosphorylation in RasV12 cells with or without MEK inhibition. The relative band densities (RD) are indicated. ( B ) (Top) Representative gel image showing RSK1, RSK2, and RSK3 cDNA levels in RasV12 cells treated with or without various RSK siRNAs. (Bottom) Representative western blot showing RSK2 expression in RasV12 cells treated with or without various RSK siRNAs. The relative band densities (RD) are indicated. Mean ± SD PpIX fluorescence in RasV12 cells ( C ) transfected with siRNA against RSKs and ( D ) treated with different concentrations of SL0101, a pan-RSK inhibitor. *p < 0.01 by one-way ANOVA with Turkey's posthoc test. ( E ) Representative western blots showing ABCB1 expression in RAS V12 cells treated with different concentrations of SL0101. p-s6 expression was used to confirm RSK inhibition, and GAPDH was used as the loading control. The relative band densities (RD) are indicated.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Expressing, Western Blot, Inhibition, Fluorescence, Transfection

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Inhibiting RSKs, ABCB1, and HIF-1α enhanced PpIX accumulation in human cancer cell lines. Human cancer cell lines DLD-1, SNB-75, Hs 578 T, and MDA MB 231 were pre-treated with or without SL0101 (RSK inhibitor), Zosuquidar (ABCB1 inhibitor), or HIF 1α inhibitor for 20 h and then with 5-ALA for 4 h. Mean ± SEM PpIX fluorescence in cell lysate compared to controls is shown. *p < 0.01 by Student's t -test.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Reduction in PpIX accumulation through the Ras/MEK-HIF-1α-FECH and Ras/MEK-RSK-ABCB1 axes in cancer cells. Ras/MEK activation increases HIF-1α expression, which in turn increases the activity of FECH, the enzyme that catalyses the conversion of PpIX to haem. Ras/MEK activation also upregulates ABCB1 expression through RSKs, which promotes the rate of PpIX efflux.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Activation Assay, Expressing, Activity Assay